EP1386925A1

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Publication number: EP1386925A1
Application number: EP02017211A
Authority: EP
Inventors: designation of the inventor has not yet been filed The
Original assignee: Girindus AG
Current assignee: Girindus AG
Priority date: 2002-07-31
Filing date: 2002-07-31
Publication date: 2004-02-04
Also published as: US20110224424A1; EP1525212B1; WO2004013154A1; US20060089494A1; AU2003250196A1; AU2003250196A8; US20100069623A1; US8304532B2; EP1525212A1

Abstract

A method for preparing an oligonucleotide comprising the steps of

a) providing a 3'-protected compound having the formula:

wherein

B is a heterocyclic base

R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylenlinkage

R₃ is a hydroxyl protecting group, a 3'-protected nucleotide or a 3'-protectedoligonucleotide

b) reacting said compound with a nucleotide derivative having a 5'-proctectiongroup in the presence of a solid supported activator to give an elongated oligonucleotidewith a P(III)-internucleotide bond

c) processing the elongated oligonucleotide with a P(III)-internucleotide bond bysteps c1) and c2) in any sequence

c1) capping by reacting with a solid supported capping agent

c2) oxidizing by reacting the oligonucleotide with a solid supported oxidizingreagent

d) removing the 5'-protection group by treatment with a solid supported agentor removing the 5'-protection group with a removal agent followed by additionof a solid supported scavenger or followed by extraction.

Description

The present invention is related to a method for preparing oligonucleotides.
The synthesis of oligonucleotides has been the subject of investigations for along period of time. Automated synthesis procedures have been developed andapparatus for the automated syntheses are commercially available. Most ofthese procedures have been developed for rather small quantities of oligonucleotides(in the range of mg). These amounts are sufficient for most investigationalpurposes.
Especially with the development of antisense therapeutics, large scale synthesisbecame a matter of considerable importance. Although relative large scaleamounts of oligonucleotides have been obtained by scale-up of solid phasesynthesis procedures, these technologies show major limitations especially highcosts for reagents and materials, e.g. the solid phase bound starting oligonucleotide.
With scale-up, the reaction time of each step of the synthesis increases.
Furthermore oligonucleotides synthesis by standard solid phase synthesis resultsin a contamination of the desired full length compound by failure sequencesarising from incomplete reaction during the synthesis cycle. At large scales thepurification of the crude oligonucleotide involves complicated isolation and chromatographicpurification of the final product.
In general synthesis methods for oligonucleotides consist of a four-step procedurefor the elongation of the oligonucleotide
1. Coupling
2. Capping
3. Oxidation
4. Deprotection of the protected hydroxyl group for the next reaction cycle.
The object of the present invention is to provide a method for the preparation ofoligonucleotides suitable for large scale (kilogram to tons) synthesis. In oneembodiment this object is solved by a liquid phase synthesis method, comprisingthe steps of
a) providing a 3'-protected compound having the formula:
wherein
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylen linkage
R₃ is a hydroxyl protecting group, a 3'-protected nucleotide or a 3'-protectedoligonucleotide
b) reacting said compound with a nucleotide derivative having a 5'-proctectiongroup in the presence of a solid supported activator to give an elongated oligonucleotidewith a P(III)-internucleotide bond
c) processing the elongated oligonucleotide with a P(III)-internucleotide bond bysteps c1) and c2) in any sequence
c1) capping by reacting with a solid supported capping agent
c2) oxidizing by reacting the oligonucleotide with a solid supported oxidizingreagent
d) removing the 5'-protection group by treatment with a solid supported agentor removing the 5' -protection group with a removal agent followed by additionof a solid supported scavenger or followed by extraction.
The method of the present invention is a solution phase synthesis wherein thereagents are solid supported. Solid supported shall cover covalently bound reagentsand reagents bound to a solid support by ionic forces.
In most cases coupling occurs of a 5'-OH-synthon with a 3'-phosphorous-synthon.Alternatively coupling of a 5'-phosphorous-synthon with a 3'-OH-synthonis also possible. Therefore in a further embodiment the invention comprisesa method comprising the steps of
a) providing a 5'-protected compound having the formula:
wherein
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylen linkage
R₅ is a hydroxyl protecting group, a 5'-protected nucleotide or a 5'-protectedoligonucleotide
b) reacting said compound with a nucleotide derivative having a 3'-proctectiongroup in the presence of a solid supported activator to give an elongated oligonucleotidewith a P(III)-internucleotide bond
c) processing the elongated oligonucleotide with a P(III)-internucleotide bond bysteps c1) and c2) in any sequence
c1) capping by reacting with a solid supported activated capping agent
c2) oxidizing by reacting the oligonucleotide with a solid supported oxidizingreagent
d) removing the 3'-protection group by treatment with a solid supported agentor removing the 3'-proctection group with a removal agent followed by additionof a solid supported scavenger or followed by extraction.In further embodiments, it is possible to couple a 3'-phosphorous synthon witha 3'-OH synthon to form a non-natural 3'-3'-internucleosidic linkage. For thesynthesis of non-natural 5'-5'-internucleosidic linkages it is possible to react a5'-phosphorous synthon with a 5'-OH synthon. These non-natural internucleosidiclinkages show increased nuclease resistance.

Step a)

B, the heterocyclic base can be a natural nucleobase or a modified one includinga non-base residue. The natural nucleobasis are adenine, guanine, thymine,cytosine and uracil. In general these bases need protection groups during thesynthesis. Suitable protected nucleobases are known to person skilled in the artfor example N-4-benzoylcytosine, N-6-benzoyl adenine, N-2-isobutiryl guanine,N-4-acetyl or isobutiryl cytosine, N-6-phenoxyacetyl adenine, N-2-tert-butylphenoxyacetyl guanine. Suitable non-base residues include for example Hydrogen,H leading to the 1',2'-dideoxyribose (dSpacer from Glen Research) whichcan be used as linker or to mimic abasic sites in an oligonucleotide (Takeshita etal., J. Biol. Chem., 1987, 262, 10171).
A suitable protection for the 2'-hydroxyl-group include but are not limited totert-butyl dimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM), fluorophenyl-metoxypiperidinyl(FPMP).
Suitable protecting groups for the 3'-hydroxyl-group include but are not limitedto tert-butyl dimethylsilyl (TBDMS), levulinyl, benzoyle. This compound is thanreacted with a nucleotide derivative with a 3'-phosphorous-synthon. The nucleotidederivative preferably has the following formula
wherein
X is a P(III)-function
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an D-substituted alkyl, a substituted alkylamino or a C4'- O2' methylenlinkage
R₅ is a hydroxyl protecting group, a 5'-protected nucleotide or a 5'-protectedoligonucleotide.
In the second embodiment, the nucleotide derivative preferably has the followingformula
wherein
X is a P(III)-function
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'- O2' methylenlinkage
R₅ is a hydroxyl protecting group, a 5'-protected nucleotide or a 5'-protectedoligonucleotide.

Step b): The coupling step

In step b) the coupling of the nucleotide or oligonucleotides occurs. The chemistryof the reaction depends on the type of activated phosphorous compound.Several methods for coupling nucleotides are known. The most common methodsare phosphoramidite and H-phosphonate. In each of these cases the phosphoris in an activated state which allows coupling with the free hydroxyl groupof the other part.
In phosphoramidite chemistry (Beaucage et al., Tetrahedron, 1992, 48, 2223-2311:Beaucage and Caruthers in unit 3:3 of Current Protocols in Nucleic AcidChemistry, Wiley) a nucleoside or oligonucleotide-3'-O-phosphoramidite wherethe P(III) phosphorus is substituted with a dialkylamine (phosphite activatinggroup) and a phosphorus protecting group (including but not limited to 2-cyanoethyl,methyl) is reacted with 5'-hydroxyl nucleoside or oligonucleotide inpresence of an activator to create a phosphite triester internucleosidic linkage.
In H-phosphonate chemistry (Froehler, Methods in Molecular Biology. Protocolsfor oligonucleotides and analogs, Humana Press, 1993, 63-80; Strömberg andStawinski, in unit 3.4 of Current Protocols in Nucleic Acid Chemistry, Wiley) anucleoside or oligonucleotide-3 ' -O-H-phosphonate is reacted with a 5' -hydroxylnucleoside or oligonucleotide in presence of an activator to create a H-phosphonatediester internucleosidic linkage.
Suitable activators for the coupling step in phosphoramidite chemistry is a solidsupport bearing a pyridinium salt, for example a solid support covalently linkedto pyridine e.g. poly(vinyl)-pyridinium or the pyridinium is a counter ion of acation exchange solid support. The cation exchange support can be a strong or aweak exchanger, for example a sulfonic acid or a carboxylic acid. The pyridiniumsalt can also be a substituted pyridinium salt, for example dichloropyridinium. Itcan further be a solid support bearing an optionally substituted azole (imidazole,triazole, tetrazole), or is the salt of a weak base anion exchange resin with astrong acid, or a weak cation exchange resin (carboxylic) in its protonated form(see US patent 5,574,146), or a solid support bearing an optionally substitutedphenol (see W. Dabkowski and al., Tet Lett, 2000, 41, 7535-7539).
For the H-phosphonate method the suitable activators are solid supports bearinga carboxylic acid chloride, sulfonic acid chloride, a chloroformate, a chlorosulfiteor a phosphorochloridate or the respective Br-compounds. Further compoundsare disclosed in WO 01/64702 A1, page 6, line 36 to page 8, line 5, incorporatedby reference and CB reese and Q song, Nucleic Acid Res., 1999, 27, 963-971.

Step c) Capping and Oxidising

Capping is understood as a reaction wherein a reagent reacts with remainingprotected compounds of step a). As the capping agent is solid supported, the 3'-protectedcompound can be removed together with the solid supported cappingagent.
For the capping step suitable reagents as capping agents are activated acids forexample carboxylic acid, chloride or sulfonic acid chloride, carboxylic acid bromide,azolide, substituted azolide, anhydride or chloroformate or phosphorochloridate,or a solid supported phosphoramidate, or a solid supported H-phosphonatemonoester. The acid group is preferably an acid group covalentlybound to a solid support commercially available cationic exchanger resins can beused as a starting material for synthesizing the solid supported carboxylic acidsor sulfonic acids.
The oxidizing reaction is used to oxidize the P(III)-internucleotide bond to aP(V)-internucleotide bond. Capping can be performed before oxidizing and viceversa. Depending on the reagents capping and oxidizing may also be combinedin one step.
For the oxidizing step the oxidizing reagent can be any oxidizing reagent usedfor prior art solid phases, but in the form of solid supported agent, either covalentlybound or bound by ionic forces. Suitable reagents are solid supportedperiodates, permanganates, osmium tetroxides, dichromates, hydroperoxides,substituted alkylamine oxides, percarboxylic acid and persulfonic acid.
These compounds are negatively charged, therefore they can be solid supportedby a suitable ion exchanger for example an ion exchanger bearing ammoniumgroups. These substances could be bound to solid support consisting for exampleof an amino, alkyl amino, dialkyl amino or trialkyl amino anion exchanger.
In oligonucleotides synthesis for investigational purposes and especially for antisensetherapeutics phosphorthioate analogs are used. In this case the oxidizingis a sulfurization. As a solid supported oxidizing reagent a solid supported sulfurizationreagent is used, for example a solid supported tetrathionate, a solidsupported alkyl or aryl sulfonyl disulfide, a solid supported optionally substituteddibenzoyl tetrasulfide, a solid supported bis(akyloxythiocarbonyl)tetrasulfide, asolid supported optionally substituted phenylacetyl disulfide, a solid supported N-[(alkylor aryl)sulfanyl] alkyl or aryl substituted succinimide and a solid supported(2-pyridinyldithio) alkyl or aryl.

Step d) Deprotection

As a 5'-protection group suitable groups are trityl groups, preferably a dimethoxytritylgroup (DMTr) or a monomethoxytrityl group (MMTr). These protectiongroups are used in conventional prior art solid phase oligonucleotidessynthesis. Other suitable 5'-protection groups are include but are not limited totert-butyl dimethylsilyl (TBDMS), levulinyl, benzoyle, fluorenemethoxycarbonyl(FMOC), the 9-phenylthioxanthen-9-yl (S-pixyl).
In the second embodiment, in step d) the 3'-protection group is removed. Suitable3'-protection groups are3'-O-tert butyl dimethyl silyl (TBDMS), 3'-O-acetate,3'-O-levulinyl groups. They can be removed by a solid-supported ammoniumfluoride, solid-supported ammonium hydroxide or solid-supported hydrazine.
In step d) of the first embodiment, the 5'-protection group is removed. Thereafterthe oligonucleotides can either be used or the oligonucleotide correspondsto the 3'-protected compound of step a) to repeat the cycle.
The use of solid supported reagents for the removal of the DMTr-protectiongroup for a completely synthesized oligonucleotide has already been reported inUS 5,808,042. The content of this document is incorporated by reference. Surprisinglythe methods disclosed in US 5,808,042 can also be applied in a solutionphase synthesis as described in the present application.
In step d) of the second embodiment, the 3'-protection group is removed.Thereafter the oligonucleotides can either be used or the oligonucleotide correspondsto the 3'-protected compound of step a) to repeat the cycle.

Step e): Repetition

In most cases the methods will be repeated at least once. When starting frommonomeric oligonucleotides the method of the present invention will result in adimer. Repeating the method of the present invention will elongate the dimer toa trimer. By repeating the method of the invention several times n-mers can besynthesized.
As the yield of a synthesis is not 100%, the overall yield of correct oligonucleotidesdecreases with the number of cycles. Depending on the yield of a singlecycle, oligonucleotides can be synthesized of up to 100 nucleotides in sufficientyield. Longer oligonucleotides are also possible.
For most cases oligonucleotides having that size will not be needed. An antisensetherapy oligonucleotides are normally in the range of 8-36 nucleotides,more preferably 12-30, most commonly in the range of the 16-26 nucleotides.
In contrast to prior art, convergent synthesis strategies are fully compatible withthe synthesis method of the present invention. Convergent synthesis methodsare methods wherein small oligonucleotides are synthesized first and the smalloligonucleotides are then combined for synthesizing larger blocks. By thismethod the number of coupling reactions can be significantly reduced. Therebythe overall yield of the oligonucleotide is increased.
In prior art, each synthesis of a small oligonucleotide had to start from the solidsupport bound nucleic acid which was rather expensive. Therefore convergentsynthesis strategies have not found much application in oligonucleotide synthesis.
Convergent synthesis has the further advantage, that the reaction product isessentially free of (n-1)mers. In prior art synthesis, the purification of oligonucleotideswith a length of n from oligonucleotides with a length of n-1 is themost difficult in purification of the oligonucleotide. By convergent synthesis,these (n-1)mers are nearly avoided, because larger fragments are combined.
In a preferred embodiment, the method of the present invention uses dimers ortrimers as the compounds in step a and/or b.
During the synthesis cycles, reagents are added in an solid supported form.These solid supported reagents are preferably removed after reaction after eachreaction step. Depending on the type of reagent it is in some cases possible toremove two or more of the solid supported reagents together.
As the synthesis is intended for the production of large amounts of oligonucleotidesit is preferred that the solid supported reagent is recycled. This recycling isobviously easier if the solid supported reagents are removed separately aftereach reaction.
The solid supported reagents can be removed by methods like filtration or centrifugation.Because of the ease of handling, filtration is the preferred way ofremoving the solid supported reagents.
A very preferred reagent for the sulfurization is a solid supported anion exchangeresin in complex with a tetrathionate having the formula S₄O₆, preferablya quaternary ammonium resin bearing tetrathionate as counter ion.
The invention will be further exemplified with the following examples.

Example 1

Synthesis of the dimer 5'-O-DMTr-T-T-3'-O-TBDMS cyanoethyl phosphite triester.

Coupling procedure of 5'-O-DMTr-T-3'-cyanoethyl phosphoramidite with 5'-OH-T-3'-O-TBDMSusing the DOWEX 50W X8 pyridinium form.

Analytical scale.

5'-OH-T-3'-O-TBDMS (11 mg, 32.5 mmol) and 5'-O-DMTr-T-3'-cyanoethylphosphoramidite (41 mg, 55.25 mmol, 1.7 eq) are dissolved in anhydrousacetonitrile (550 ml). The solution is transferred under argon in a NMR tubecontaining the DOWEX 50W X8 pyridinium form (100 mg, 0.30 mmol pyrH⁺,9.2 eq). The reaction is followed by³¹P NMR. Before the NMR experimentdeuterated acetonitrile (50 ml) is added. The yield is determined by³¹P NMR.After 3 h the desired dimer T-T phosphite triester is obtained with 100% ofyield compared to 5'-OH-T-3'-O-TBDMS. The crude is a mixture of 5'-O-DMTr-T-3'-cyanoethylphosphoramidite (³¹P NMR (CD₃CN) d 149.14,149.07, 14.7%),5'-O-DMTr-T-T-3'-O-TBDMS cyanoethyl phosphite triester (d 140.53, 70.2%),5'-O-DMTr-T-3'-cyanoethyl hydrogenophosphonate (d 8.74, 15.1%).

Example 2

Coupling procedure of 5'-O-DMTr-T-3'-cyanoethyl phosphoramidite with 5'-OH-T-3'-O-TBDMSusing the poly(4-vinylpyridinum p-toluenesulfonate)(Aldrich).

Analytical scale.

5'-OH-T-3'-O-TBDMS (11 mg, 32.5 mmol) and 5'-O-DMTr-T-3'-cyanoethylphosphoramidite (41 mg, 55.25 mmol, 1.7 eq) are dissolved in anhydrousacetonitrile (550 ml). The solution is transferred under argon in a NMR tubecontaining the poly(4-vinylpyridinum p-toluenesulfonate) (100 mg, 0.33 mmoltos^-, 10.3 eq). The reaction is followed by³¹P NMR. Before the NMR experimentdeuterated acetonitrile (50 ml) is added. The yield is determined by³¹PNMR. After 1 h 45 the desired dimer T-T phosphite triester is obtained with82% of yield compared to 5'-OH-T-3'-O-TBDMS. The crude is a mixture of 5'-O-DMTr-T-T-3'-O-TBDMScyanoethyl phosphite triester (³¹P NMR (CD₃CN) d140.54, 48.2%), 5'-O-DMTr-T-3'-cyanoethyl hydrogenophosphonate (d 8.77,51.8%).

Example 3

Synthesis of the dimer 5'-OH-T-T-3'-O-TBDMS cyanoethyl phosphorothioatetriester.

Coupling procedure of 5'-O-DMTr-T-3'-cyanoethyl phosphoramidite with 5'-OH-T-3'-O-TBDMSusing the DOWEX 50W X8 pyridinium form.
A solution of 5'-OH-T-3'-O-TBDMS (124 mg, 0.35 mmol) and 5'-O-DMTr-T-3'-cyanoethylphosphoramidite (441 mg, 0.59 mmol, 1.7 eq) in anhydrous acetonitrile(6 ml) is added to DOWEX 50W X8 pyridinium form (1 g, 3 mmol pyrH⁺,9.5 eq). The resulting mixture is shaken for 4 h 45. The reaction is followed by³¹P NMR and the yield is also determined by³¹P NMR. The desired dimer 5'-O-DMTr-T-T-3'-O-TBDMS cyanoethyl phosphite triester is obtained with 100% ofyield compared to 5'-OH-T-3'-O-TBDMS. The crude is a mixture of 5'-O-DMTr-T-3'-cyanoethylphosphoramidite (³¹P NMR (CD₃CN) d 149.17,149.10, 5.4%),5'-O-DMTr-T-T-3'-O-TBDMS cyanoethyl phosphite triester (d 140.57, 140.54,68.3%), 5'-O-DMTr-T-3'-cyanoethyl hydrogenophosphonate (d 8.75, 8.71,26.3%).
Sulfurization: The DOWEX 50W X8 resin is filtered off and the resulting solutionis added to AMBERLYST A26 tetrathionate form (1.44 g, 2.44 mmol S₄O₆^2-,7 eq.). The reaction is followed by³¹P NMR and the yield is also determined by³¹P NMR. After 20 h the desired dimer 5'-O-DMTr-T-T-3'-O-TBDMS cyanoethylphosphorothioate triester is obtained with 97% of yield. The crude is a mixtureof 5'-O-DMTr-T-3'- cyanoethyl thiophosphoramidate (³¹P NMR (CD₃CN) d71.16, 4.0%), 5'-O-DMTr-T-T-3'-O-TBDMS cyanoethyl phosphorothioate triester(d 68.28, 68.23, 69.5%), 5'-O-DMTr-T-3'-cyanoethyl hydrogenophosphonate(d 8.75, 8.71, 26.5%). MALDI-TOF MS (negative mode, trihydroxyacetophenoneas matrix) ammonia treatment of an aliquot gives 5'-OH-T-T- 3'-O-TBDMSphosphorothioate diester: [M-H]^- m/z_exp = 978.12, m/z_calc= 977.13.
Detritylation: The AMBERLYST A26 is filtered off and the solvent are evaporated.The crude is dissolved in 4ml of CH₂Cl₂/CH₃OH (7/3) and cooled in anice bath. To this solution is added 1 ml of a solution of benzene sulfonic acid10% in CH₂Cl₂/CH₃OH (7/3). The solution is stirred 15 min at 0°C. The reactionis washed with 10 ml of a saturated solution of NaHCO₃, the organic layer isseparated, dried (Na₂SO₄), evaporated, and purified on a silica gel column. Thedesired dimer T-T is eluted with CH₂Cl₂/CH₃OH (95/5). The appropriates fractionsare collected and evaporated to give 230 mg of a white foam in a yield of83% compared to 5'-OH-T-3'-O-TBDMS.³¹P NMR (CD₃CN) d 68.29, 68.19.MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp = 730.46, m/z_calc = 730.82. The spectrophotometric purity (97%) isdetermined by HPLC at 260 nm.

Example 4

Synthesis of the trimer 5'-OH-T-T-T-3'-O-TBDMS cyanoethyl phosphorothioatetriester.

Coupling procedure of 5'-O-DMTr-T-3'-cyanoethyl phosphoramidite with thedimer 5'-OH-T-T-3'-O-TBDMS cyanoethyl phosphorothioate triester using theDOWEX 50W X8 pyridinium form.
A solution of 5'-OH-T-T-3'-O-TBDMS cyanoethyl phosphorothioate triester(230 mg, 0.31 mmol) and 5'-O-DMTr-T-3'-cyanoethyl phosphoramidite (399mg, 0.54 mmol, 1.7 eq) in anhydrous acetonitrile (8 ml) is added to DOWEX50W X8 pyridinium form (1 g, 3 mmol pyrH⁺, 9.5 eq). The resulting mixture isshaken for 5 h. The reaction is followed by³¹P NMR and the yield is also determinedby³¹P NMR. The desired trimer 5'-O-DMTr-T-T-T-3'-O-TBDMS cyanoethylphosphite triester is obtained with 100% of yield compared to the dimer5'-OH-T-T-3'-O-TBDMS cyanoethyl phosphorothioate triester. The crude is amixture of 5'-O-DMTr-T-3'-cyanoethyl phosphoramidite (³¹P NMR (CD₃CN) d149.16,149.10, 17.7%), 5'-O-DMTr-T-T-T-3'-O-TBDMS cyanoethyl phosphitetriester (d 140.85, 140.68, 140.37, 140.30, d 68.07, 68.02, 68.3%), 5'-O-DMTr-T-3'-cyanoethylhydrogenophosphonate (d 8.7, 8.68, 14%).
Sulfurization: The DOWEX 50W X8 pyridinium form is filtered off and theresulting solution is added to AMBERLYST A26 tetrathionate form (1.3 g, 2.44mmol S₄O₆^2-, 7 eq.). The reaction is followed by³¹P NMR and the yield is alsodetermined by³¹P NMR. After 45 h the desired trimer 5'-O-DMTr-T-T-T-3'-O-TBDMScyanoethyl phosphorothioate triester is obtained with 100% of yield.MALDI-TOF MS (negative mode, trihydroxyacetophenone as matrix) [M-H]^-m/z_exp = 1297.89, m/z_calc = 1296.38 after 30 min of ammonia treatment toremove the cyanoethyl protecting group. The crude is a mixture of 5'-O-DMTr-T-3'-cyanoethyl thiophosphoramidate (³¹P NMR (CD₃CN) d 72.04, 71.17,14.0%), 5'-O-DMTr-T-T-T-3'-O-TBDMS cyanoethyl phosphorothioate triester(d 68.17, 68.12, 68.07, 67.96, 67.80, 67.58, 73.8%), 5'-O-DMTr-T-3'-cyanoethylhydrogenophosphonate (d 8.76, 8.71, 12.2%).
Detritylation: The AMBERLYST A26 is filtered off and the solvent are evaporated.The crude is dissolved in 4ml of CH₂Cl₂/CH₃OH (7/3) and cooled in anice bath. To this solution is added 1 ml of a solution of benzene sulfonic acid10% in CH₂Cl₂/CH₃OH (7/3). The solution is stirred 45 min at 0°C. The reactionis washed with 10 ml of a saturated solution of NaHCO₃, the organic layer isseparated, dried (Na₂SO₄), evaporated, and purified on a silica gel column. Thedesired trimer T-T-T is eluted with CH₂Cl₂/CH₃OH (95/5). The appropriatesfractions are collected and evaporated to give 221 mg of a white foam in ayield of 63% compared to the dimer 5'-OH-T-T-3'-O-TBDMS cyanoethyl phosphorothioatetriester.³¹P NMR (CD₃CN) d 68.53, 68.38, 68.34, 67.74, 67.54.MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp = 1103.91, m/z_calc = 1104.15. The spectrophotometric purity (93%) isdetermined by HPLC at 260 nm.

Example 5

Synthesis of the dimer 5'-OH-T-dA^Bz-3'-O-TBDMS cyanoethyl phosphorothioatetriester.

Coupling procedure of 5'-O-DMTr-T-3'-phosphoramidite with 5'-OH-dA^BZ-3'-O-TBDMSusing the poly(4-vinylpyridinump-toluenesulfonate) (Aldrich).
A solution of 5'-OH-dA^Bz-3'-O-TBDMS (176 mg, 0.38 mmol) and 5'-O-DMTr-T-3'-cyanoethylphosphoramidite (560 mg, 0.75 mmol, 2 eq) in anhydrous acetonitrile(6 ml) is added to poly(4-vinylpyridinump-toluenesulfonate) (1.15 g,3.84 mmol tos^-, 10.2 eq). The resulting mixture is shaken for 4 h 30 min. Thereaction is followed by³¹P NMR and the yield is also determined by³¹P NMR.The desired dimer 5'-O-DMTr-T-dA^Bz-3'-O-TBDMS cyanoethyl phosphite triesteris obtained with 100 % of yield compared to the 5'-OH-dA^BZ-3'-O-TBDMS.The crude is a mixture of 5'-O-DMTr-T-3'-cyanoethyl phosphoramidite(³¹P NMR (CD₃CN) d 149.10,149.05, 12.3%), 5'-O-DMTr-T-A^Bz-3'-O-TBDMScyanoethyl phosphite triester (d 140.52, 140.37, 50%), 5'-O-DMTr-T-3'-cyanoethylhydrogenophosphonate (d 8.72, 8.69, 37.7%).
Sulfurization: The poly(4-vinylpyridinum p-toluenesulfonate) is filtered offand the resulting solution is added to AMBERLYST A26 tetrathionate form(1.55 g, 2.63 mmol S₄O₆^2-, 7 eq.). The reaction is followed by³¹P NMR. Thereaction mixture is shaken for 24 h 30. The desired dimer 5'-O-DMTr-T-dA^Bz-3'-O-TBDMScyanoethyl phosphorothioate triester is isolated after filtration ofthe resin, evaporation of the solvent, and column chromatography (silica gel;CH₂Cl₂ / MeOH (50/1)). Yield : 325 mg, 0.28 mmol, 76%.³¹P NMR (CD₃CN) d68.34, 68.15. MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix)[M+H]⁺ m/z_exp = 1144.22, m/z_calc = 1146.32.
Detritylation: The 5'-O-DMTr-T-dA^BZ- 3'-O-TBDMS cyanoethyl phosphorothioatetriester is dissolved in 10 ml of CH₂Cl₂/CH₃OH (7/3) and cooled inan ice bath. To this solution is added 1 ml of a solution of benzene sulfonicacid 10% in CH₂Cl₂/CH₃OH (7/3). The solution is stirred 35 min at 0°C. Thereaction is washed with 20 ml of a saturated solution of NaHCO₃, the organiclayer is separated, dried (Na₂SO₄), evaporated, and purified on a silica gelcolumn. The desired dimer T-dA^Bz is eluted with CH₂Cl₂/CH₃OH (95/5). Theappropriates fractions are collected and evaporated to give a white foam.Yield: 223 mg, 0.26 mmol, 71%.³¹P NMR (CD₃CN) d 68.06, 67.89. MALDI-TOFMS (positive mode, trihydroxyacetophenone as matrix) [M-H]⁺ m/z_exp =842.18, m/z_calc = 843.95; (negative mode, trihydroxyacetophenone as matrix)ammonia treatment of an aliquot gives 5'-OH-T-dA- 3'-O-TBDMS phosphorothioatediester: [M-H]^- m/z_exp = 685.38, m/z_calc = 684.77.

Example 6

Synthesis of the trimer 5'-O-DMTr-dA^Bz-T-dA^BZ-3'-O-TBDMS cyanoethyl phosphorothioatetriester.

Coupling procedure of 5'-O-DMTr-dA^Bz-3'-cyanoethyl phosphoramidite withthe dimer 5'-OH-T-A^Bz-3'-O-TBDMS phosphorothioate triester using the poly(4-vinylpyridinump-toluenesulfonate) (Aldrich).
A solution of the dimer 5'-OH-T-dA^Bz-3'-O-TBDMS phosphorothioate triester(223 mg, 0.26 mmol) and 5'-O-DMTr-dA^Bz-3'-cyanoethyl phosphoramidite (432mg, 0.50 mmol, 1.9 eq) in anhydrous acetonitrile (20 ml) is added to poly(4-vinylpyridinump-toluenesulfonate) (0.8 g, 2.7 mmol tos^-, 10.3 eq). The resultingmixture is shaken for 6 h 30. The reaction is followed by³¹P NMR andthe yield is also determined by³¹P NMR. The desired trimer 5'-O-DMTr-dA^Bz-T-dA^Bz-3'-O-TBDMScyanoethyl phosphite triester is obtained with 62% of yieldcompared to the dimer 5'-OH-T-dA^Bz-3'-O-TBDMS phosphorothioate triester.The crude is a mixture of 5'-O-DMTr-dA^Bz-3'-cyanoethyl phosphoramidite (³¹PNMR (CD₃CN) d 149.14, 8.4%), 5'-O-DMTr-dA^Bz-T-dA^Bz-3'-O-TBDMS cyanoethylphosphite triester (d 140.90, 140.77, 67.85, 67.79, 43.3%), 5'-OH-T-dA^Bz-3'-O-TBDMSphosphorothioate triester (d 68.03, 67.89, 13.4%), 5'-O-DMTr-dA^Bz-3'-cyanoethylhydrogenophosphonate (d 8.71,8.66, 34.9%).
Sulfurization: The poly(4-vinylpyridinum p-toluenesulfonate) is filtered offand the resulting solution is added to AMBERLYST A26 tetrathionate form(0.78 g, 1.33 mmol S₄O₆^2-, 5 eq.). The reaction is followed by³¹P NMR and theyield is also determined by³¹P NMR. After 14 h 30 the desired trimer 5'-O-DMTr-dA^Bz-T-dAg^Bz-3'-O-TBDMScyanoethyl phosphorothioate triester is obtainedwith 100% of yield. The crude is a mixture of 5'-O-DMTr-dA^Bz-3'- cyanoethylthiophosphoramidate (³¹P NMR (CD₃CN) d 71.88, 71.21, 10%), 5'-O-DMTr-dA^Bz-T-dA^Bz-3'-O-TBDMScyanoethyl phosphorothioate triester (25.9%)and 5'-OH-T-dA^Bz-3'-O-TBDMS cyanoethyl phosphorothioate triester (16.2%)(d 68.08, 68.05, 67.93, 67.89, 67.85, 67.79, 67.57), 5'-O-DMTr-T-3'-cyanoethylphosphorothioate diester (d 57.38, 4.8%), 5'-O-DMTr-dA^Bz-3'-cyanoethylhydrogenophosphonate (d 8.75,8.70, 43.11%). MALDI-TOF MS(positive mode, trihydroxyacetophenone as matrix) [M+H]⁺ m/z_exp = 1631.68,m/z_calc = 1632.77; (negative mode, trihydroxyacetophenone as matrix) ammoniatreatment of an aliquot gives 5'-OH-T-dA- 3'-O-TBDMS phosphorothioatediester: [M-H]- m/z_exp = 1316.45, m/z_calc= 1316.43.

Example 7

Synthesis of the dimer 5'-O-DMTr-dC^Bz-T-3'-O-Lev cyanoethyl phosphite triester.

Coupling procedure of 5'-O-DMTr-dC^Bz-3'-cyanoethyl phosphoramidite with5'-OH-T-3'-O-Lev using the DOWEX 50W X8 pyridinium form.

Analytical scale.

5'-OH-T-3'-O-Lev (20 mg, 58.9 mmol) and 5'-O-DMTr-dC^Bz-3'-cyanoethylphosphoramidite (83.4 mg, 100 mmol, 1.7 eq) are dissolved in anhydrousacetonitrile (550 ml). The solution is transferred under argon in a NMR tubecontaining the DOWEX 50W X8 pyridinium form (181 mg, 0.54 mmol pyrH⁺,9.2 eq). The reaction is followed by³¹P NMR. Before the NMR experimentdeuterated acetonitrile (50 ml) is added. The yield is determined by³¹P NMR.After 6 h the desired dimer T-dC^Bz phosphite triester is obtained with 100% ofyield compared to 5'-OH-T-3'-O-Lev. The crude is a mixture of 5'-O-DMTr-dC^Bz-3'-cyanoethylphosphoramidite (³¹P NMR (CD₃CN) d 149.36,149.32,11%), 5'-O-DMTr-T-dC^Bz-3'-O-TBDMS cyanoethyl phosphite triester (d140.52, 140.39, 70%), 5'-O-DMTr- dC^Bz -3'-cyanoethyl hydrogenophosphonate(d 8.90, 8.58, 19%).

Example 8

Synthesis of the dimer 5'-OH-dC^Bz-T-3'-O-Lev cyanoethyl phosphorothioatetriester.

Coupling procedure of 5'-O-DMTr-dC^Bz-3'-cyanoethyl phosphoramidite with5'-OH-T-3'-O-Lev using the DOWEX 50W X8 pyridinium form.
A solution of 5'-OH-T-3'-O-Lev (119 mg, 0.35 mmol) and 5'-O-DMTr-dC^Bz-3'-cyanoethylphosphoramidite (496 mg, 0.60 mmol, 1.7 eq) in anhydrous acetonitrile(10 ml) is added to DOWEX 50W X8 pyridinium form (1,1 g, 3,3 mmolpyrH⁺, 9.4 eq). The resulting mixture is shaken for 5 h 30 min. The reaction isfollowed by³¹P NMR and the yield is also determined by³¹P NMR. The desired dimer 5'-O-DMTr-dC^Bz-T-3'-O-Lev cyanoethyl phosphite triester is obtainedwith 100% of yield compared to 5'-OH-T-3'-O-Lev. The crude is a mixture of5'-O-DMTr-T-dC^Bz-3'-O-Lev cyanoethyl phosphite triester (³¹P NMR (CD₃CN) d140.59, 140.45, 64%), 5'-O-DMTr-dC^Bz-3'-cyanoethyl hydrogenophosphonate(d 8.68, 8.66, 36%).
Sulfurization: The DOWEX 50W X8 resin is filtered off and the resulting solutionis added to AMBERLYST A26 tetrathionate form (1.44 g, 2.44 mmol S₄O₆^2-,7 eq.). The reaction is followed by³¹P NMR. The reaction mixture is shaken for16 h. The desired dimer 5'-O-DMTr-dC^Bz-T-3'-O-Lev cyanoethyl phosphorothioatetriester is isolated after filtration of the resin, evaporation of thesolvent and column chromatography (silica gel; CH₂Cl₂ / MeOH (97/3)). Thecrude is a mixture of 5'-O-DMTr-T-dC^Bz-3'-O-Lev cyanoethyl phosphorothioatetriester (³¹P NMR (CD₃CN) d 68.05, 67.89, 83.7%), 5'-O-DMTr-dC^Bz-3'-cyanoethylhydrogenophosphonate (d 8.63, 16.3%). The spectrophotometricpurity determined by HPLC at 260 nm is 80%.

Detrytilation of the dimer 5'-O-DMTr-dC^Bz-T-3'-O-Lev cyanoethylphosphorothioate triester with the DOWEX 50 W X8 H⁺ form (Aldrich).

To the mixture of the dimer 5'-O-DMTr-dC^Bz-T-3'-O-Lev cyanoethyl phosphorothioatetriester (121 mmol estimated) and 5'-O-DMTr-T-3'-cyanoethylhydrogenophosphonate diester (44 mmol estimated) in solution in 10 ml ofCH₂Cl₂/MeOH (7/3) is added the DOWEX 50 W X8 H⁺ form (1.4 g, 7 mmol H⁺,58 eq / dimer). The reaction is followed by reverse phase HPLC. After 15 minthe detritylation is complete. The resin is filtered off and the solvents areevaporated. The desired dimer 5'-OH-dC^Bz-T-3'-O-Lev cyanoethyl phosphorothioatetriester is purified by precipitation from CH₂Cl₂/MeOH (9/1) indiethylether.³¹P NMR (CD₃OD) d 68.24, 67.90, MALDI-TOF MS (positive mode,trihydroxyacetophenone as matrix) [M-H]⁺ m/z_exp = 803.11 m/z_calc = 803.76.The spectrophotometric purity (97 %) is determined by HPLC at 260 nm.

Example 9

Synthesis of the dimer 5'-OH-T-T-3'-O-Lev cyanoethyl phosphorothioate triester.

Coupling procedure of 5'-O-DMTr-T-3'-cyanoethyl phosphoramidite with 5'-OH-T-3'-O-Levusing the DOWEX 50W X8 pyridinium form.
A solution of 5'-OH-T-3'-O-Lev (100 mg, 0.29 mmol) and 5'-O-DMTr-T-3'-cyanoethylphosphoramidite (547 mg, 0.73 mmol, 2.5 eq) in anhydrous acetonitrile(10 ml) is added to DOWEX 50W X8 pyridinium form (0.9 g, 2.7 mmolpyrH⁺, 9.3 eq). The resulting mixture is shaken for 10 h. The reaction is followedby³¹P NMR and by reverse phase HPLC. The excess of 5'-O-DMTr-T-3'-cyanoethylphosphoramidite is hydrolysed with 500 ml of water The desireddimer 5'-O-DMTr-T-T-3'-O-Lev cyanoethyl phosphite triester is obtained with100% of yield compared to 5'-OH-T-3'-O-Lev. The crude is a mixture of 5'-O-DMTr-T-T-3'-O-Levcyanoethyl phosphite triester (HPLC % Area = 55%) and5'-O-DMTr-T-3'-cyanoethyl hydrogenophosphonate (HPLC % Area = 45%) .
Sulfurization: The DOWEX 50W X8 resin is filtered off and the resulting solutionis added to AMBERLYST A26 tetrathionate form (0.8 g, 1.5 mmol S₄O₆^2-, 5eq.). The reaction is followed by³¹P NMR and by reverse phase HPLC. After 15h the desired dimer 5'-O-DMTr-T-T-3'-O-Lev cyanoethyl phosphorothioatetriester is obtained with 100% of yield. The crude is a mixture of 5'-O-DMTr-T-T-3'-O-Levcyanoethyl phosphorothioate triester (³¹P NMR d 68.04, HPLC %Area = 57%), 5'-O-DMTr-T-3'-cyanoethyl hydrogenophosphonate (d 8.77,HPLC % Area = 43%).

Detrytilation of the dimer 5'-O-DMTr-T-T-3'-O-Lev cyanoethyl phosphorothioatetriester with the DOWEX 50 W X8 H⁺ form (Aldrich).

The AMBERLYST A26 resin is filtered off and the solvents are evaporated. Tothe mixture of the dimer 5'-O-DMTr-T-T-3'-O-Lev cyanoethyl phosphorothioatetriester (0.29 mmol estimated) and 5'-O-DMTr-T-3'-cyanoethyl hydrogeno-phosphonate(0.22 mmol estimated) in solution in 20 ml of CH₂Cl₂/MeOH (7/3)is added the DOWEX 50 W X8 H⁺ form (3.7 g, 18.5 mmol H⁺, 64 eq / dimer).
The reaction is followed by reverse phase HPLC. After 30 min the detritylationof the dimer is complete. The resin is filtered off and the solvents are evaporated.The desired dimer 5'-OH-T-T-3'-O-Lev cyanoethyl phosphorothioatetriester is purified by precipitation from CH₂Cl₂/MeOH (9/1) in diethylether.³¹PNMR (CD₃CN) d 67.88, 67.73. MALDI-TOF MS (positive mode, trihydroxyacetophenoneas matrix) [M-H]⁺ m/z_exp = 713.79 m/z_calc = 714.66. The purity(95%) is determined by HPLC.

Example 10

Synthesis of dimer 5'-O-DMTr-dA^Bz-dA^Bz-3'-O-TBDMS cyanoethyl phosphorothioatetriester dimer.

Coupling procedure of 5'-O-DMTr-dA^Bz-3'-cyanoethyl-phosphoramidite with5'-OH-dA^Bz-3'-O-TBDMS using the DOWEX 50W X8 pyridinium form:
5'-OH-dA^Bz-3'-O-TBDMS (100 mg, 0.21 mmol) and 5'-O-DMTr-dA^Bz-3'-cyanoethyl-phosphoramidite(311 mg, 0.36 mmol, 1.7 eq) are dissolved inanhydrous acetonitrile (15 ml). The solution is transferred under argon in aflask containing the DOWEX 50W X8 pyridinium form (655 mg, 1.97 mmolpyrH⁺, 9.2 eq) and is shaken for 4 h 30 min. The reaction is followed by³¹PNMR. The yield is determined by³¹P NMR. The desired dA-dA phosphite triesterdimer is obtained with 92% of yield compared to the 5'-OH-dA^Bz-3'-O-TBDMS.The crude is a mixture of 5'-O-DMTr-dA^Bz-3'-cyanoethyl phosphoramidite (³¹PNMR (CD₃CN) d 149.25, 149.13; 27.7%), 5'-O-DMTr-dA^Bz-dA^Bz-3'-O-TBDMScyanoethyl phosphite triester (d 140.75, 140.38; 53.9%), 5'-O-DMTr-dA^Bz-3'-cyanoethylhydrogenophosphonate (d 8.69, 8.64; 18.5%).
Sulfurization: To a solution of 5'-O-DMTr-dA^Bz-dA^Bz-3'-O-TBDMS phosphitetriester dimer (0.2 mmol) in anhydrous acetonitrile is added AMBERLYST A26tetrathionate form (5.4 eq., 1.14 mmol S₄O₆^2-, 0.63 g). The reaction mixture isshaken for 20 h. The reaction is followed by³¹P NMR. The yield is determinedby³¹P NMR. After filtration of the resins the desired dimer dA-dA phosphorothioatetriester is obtained with 88% of yield compared to the 5'-OH-dA^Bz-3'-O-TBDMS. The crude is a mixture of 5'-O-DMTr-dA^Bz-3'-cyanoethylthiophosphoramidate (³¹P NMR (CD₃CN) d 71.85, 71.22; 29.0%), 5'-O-DMTr-dA^Bz-dA^Bz-3'-O-TBDMScyanoethyl phosphorothioate (d 68.08, 68.01; 51.5%),5'-O-DMTr-dA^Bz-3'-cyanoethyl hydrogenophosphonate (d 8.66, 8.59; 19.5%).

Example 11

Synthesis of the dimer 5'-OH-dC^Bz-dA^Bz-3'-O-TBDMS cyanoethyl phosphorothioatetriester.

Coupling procedure of 5'-O-DMTr-dC^Bz-3'-cyanoethyl-phosphoramidite with5'-OH-dA^Bz-3'-O-TBDMS using the poly(4-vinylpyridinum p-toluenesulfonate)(Aldrich):
5'-OH-dA^Bz-3'-O-TBDMS adenosine (100 mg, 0.21 mmol) and 5'-O-DMTr-dC^Bz-3'-cyanoethyl-phosphoramidite(365 mg, 0.43 mmol, 2. eq) are dissolved inanhydrous acetonitrile (15 ml). The solution is transferred under argon in aflask containing the poly(4-vinylpyridinum p-toluenesulfonate) (655 mg, 2.17mmol tos^-, 10.2 eq) and is shaken for 5 h 50 min. The reaction is followed by³¹P NMR. The yield is determined by³¹P NMR. The desired dC-dA phosphitetriester dimer is obtained with 100% of yield compared to the 5'-OH-dA^Bz-3'-O-TBDMS.The crude is a mixture of 5'-O-DMTr-dC^Bz-dA^BZ-3'-O-TBDMS cyanoethylphosphite triester (³¹P NMR (CD₃CN) (d 140.55, 140.49; 53.9%), 5'-O-DMTr-dC^Bz-3'-cyanoethylhydrogenophosphonate (d 8.67; 18.5%).
Sulfurization: To a solution of 5'-O-DMTr-dC^Bz-dA^Bz-3'-O-TBDMS cyanoethylphosphite triester dimer (0.21 mmol) in anhydrous dichloromethane is addedAMBERLYST A26 tetrathionate form (0.63 g, 1.14 mmol S₄O₆^2-, 5.3 eq.). Thereaction mixture is shaken for 14 h 30 min. The reaction is followed by³¹PNMR. The yield is determined by³¹P NMR. After filtration of the resins the desireddC-dA phosphorothioate triester dimer is obtained with 100% of yieldcompared to the 5'-OH-dA^Bz-3'-O-TBDMS. The crude is a mixture of 5'-O-DMTr-dC^Bz-dA^Bz-3'-O-TBDMScyanoethyl phosphorothioate (³¹P NMR (CD₃CN)(d 68.14, 68.07; 50.6%), 5'-O-DMTr-dC^Bz-3'-cyanoethyl hydrogenophosphonate (d 8.67; 49.4%). Purification is attempted on a silica gel column,which is treated with triethylamine. Chromatography leads to complete loss ofthe cyanoethyl group. The dC^Bz-dA^Bz phosphorothioate dimer is eluted withCH₂Cl₂/CH₃OH (80/1). The appropriates fractions are collected and evaporatedto give a colorless oil. Yield: 185 mg, 0.14 mmol, 68%;³¹P NMR (CD₃CN) d57.58, 57.45; MALDI-TOF MS (positive mode, trihydroxyacetophenone asmatrix) [M-DMTr+2H]⁺ m/z_exp = 879.42, m/z_calc = 878.97.

Example 12

Synthesis of the dimer 5'-OH-dA^Bz-dA^Bz-3'-O-TBDMS cyanoethyl phosphorothioatetriester.

Coupling procedure of 5'-O-DMTr-dA^Bz-3'-cyanoethyl-phosphoramidite with5'-OH-dA^Bz-3'-O-TBDMS using the poly(4-vinylpyridinum p-toluenesulfonate)(Aldrich):
5'-OH-dA^Bz-3'-O-TBDMS (102 mg, 0.22 mmol) and 5'-O-DMTr-dA^Bz-3'-cyanoethyl-phosphoramidite(381 mg, 0.44 mmol, 2.05 eq) are dissolved inanhydrous dichloromethane (15 ml). The solution is transferred under argon ina flask containing the poly(4-vinylpyridinum p-toluenesulfonate) (655 mg, 2.19mmol tos^-, 10.1 eq) and is shaken for 5 h 40 min. The reaction is followed by³¹P NMR. The yield is determined by³¹P NMR. The desired dA-dA phosphitetriester dimer is obtained with 100% of yield compared to the 5'-OH-dA^Bz-3'-O-TBDMS.The crude is a mixture of 5'-O-DMTr-dA^Bz-dA^Bz-3'-O-TBDMS cyanoethylphosphite triester (³¹P NMR (CD₃CN) (d 140.77, 140.46; 66.9%), 5'-O-DMTr-dA^Bz-3'-cyanoethylhydrogenophosphonate (d 8.50, 8.41; 33.1%).
Sulfurization: To a solution of 5'-O-DMTr-dA^Bz-dA^Bz-3'-O-TBDMS cyanoethylphosphite triester dimer (0.22 mmol) in anhydrous dichloromethane is addedAMBERLYST A26 tetrathionate form (0.87 g, 1.14 mmol S₄O₆^2-, 5.4 eq.). Thereaction mixture is shaken for 22 h. The reaction is followed by³¹P NMR. Theyield is determined by³¹P NMR. After filtration of the resins the desired dA-dAphosphorothioate triester dimer is obtained with 100 % of yield compared to the 5'-OH-A^Bz-3'-O-TBDMS. The crude is a mixture of 5'-O-DMTr-dA^Bz-dA^Bz-3'-O-TBDMScyanoethyl phosphorothioate (³¹P NMR (CD₃CN) (d 68.17, 67.89;62.3%), 5'-O-DMTr-dA^Bz-3'-cyanoethyl hydrogenophosphonate (d 8.45, 8.35;37.7%).
Detritylation: To a solution of 5'-O-DMTr-dA^Bz-dA^Bz-3'-O-TBDMS cyanoethylphosphorothioate triester (0.22 mmol) in 10 ml CH₂Cl₂/CH₃OH (7/3) is added0.63 ml (0.3 mmol, 1.4 eq.) of a solution of benzene sulfonic acid 10% inCH₂Cl₂/CH₃OH (7/3). The solution is stirred 45 min at 0°C. The reaction iswashed with 10 ml of a saturated solution of NaHCO₃, the organic layer isseparated, dried (Na₂SO₄), evaporated, and purified on a silica gel column. Thedesired. dA-dA dimer is eluted with CH₂Cl₂/CH₃OH (33/1). The appropriatesfractions are collected and evaporated to give a colorless oil. Yield: 73 mg, 76mmol, 35%;³¹P NMR (CD₃CN) d 67.80, 67.71; MALDI-TOF MS (positive mode,trihydroxyacetophenone as matrix) [M-H]⁺ m/z_exp = 957.01, m/z_calc = 957.07;HPLC (spectrophotometrical purity at 260 nm = 95 %).

Example 13

Synthesis of the dimer 5'-OH-dG^iBu-dA^Bz-3'-O-TBDMS cyanoethyl phosphorothioatetriester.

Coupling procedure of 5'-O-DMTr-dG^iBu-3'-cyanoethyl-phosphoramidite with5'-OH-dA^Bz-3'-O-TBDMS using the poly(4-vinylpyridinump-toluenesulfonate)(Aldrich):
5'-OH-dA-3'-O-TBDMS (100 mg, 0.21 mmol) and 5'-O-DMTr-dG^iBu-3'-cyanoethyl-phosphoramidite(352 mg, 0.42 mmol, 1.97 eq) are dissolved inanhydrous acetonitrile (20 ml). The solution is transferred under argon in aflask containing the poly(4-vinylpyridinum p-toluenesulfonate) (655 mg, 2.19mmol tos^-, 10.3 eq) and is shaken for 5 h 30 min. The reaction is followed by³¹P NMR. The yield is determined by³¹P NMR. The desired dG-dA phosphitetriester dimer is obtained with 100% of yield compared to the 5'-OH-dA^Bz-3'-O-TBDMS.The crude is a mixture of 5'-O-DMTr-dG^iBu-dA^Bz-3'-O-TBDMS cyanoethyl phosphite triester (³¹P NMR (CD₃CN) (d 140.65, 140.45; 51.2%), 5'-O-DMTr-dG^iBu-3'-cyanoethylhydrogenophosphonate (d 9.00, 8.81; 48.8%).
Sulfurization: To a solution of 5'-O-DMTr-dG^iBu-dA^Bz-3'-O-TBDMS cyanoethylphosphite triester dimer (0.21 mmol) in anhydrous acetonitrile is addedAMBERLYST A26 tetrathionate form (0.63 g, 1.14 mmol S₄O₆^2-, 5.4 eq.). Thereaction mixture is shaken for 2 h. The reaction is followed by³¹P NMR. Theyield is determined by³¹P NMR. After filtration of the resins the desired dG-dAphosphorothioate triester dimer is obtained with 100% of yield compared tothe 5'-OH-dABZ-3'-O-TBDMS. The crude is a mixture of 5'-O-DMTr-dG^iBu-dA^Bz-3'-O-TBDMScyanoethyl phosphorothioate (³¹P NMR (CD₃CN) (d 68.15, 68.02;50.4%), 5'-O-DMTr-dG^iBu-3'-cyanoethyl hydrogenophosphonate (d 8.91, 8.68;49.6%).
Detritylation: To a solution of 5'-O-DMTr-dG^iBu-dA^Bz-3'-O-TBDMS cyanoethylphosphorothioate triester (0.21 mmol) in 10 ml CH₂Cl₂/CH₃OH (7/3) is added0.5 ml (0.3 mmol, 1.4 eq.) of a solution of benzene sulfonic acid 10% inCH₂Cl₂/CH₃OH (7/3). The solution is stirred 20 min at 0°C. The reaction iswashed with 10 ml of a saturated solution of NaHCO₃, the organic layer isseparated, dried (Na₂SO₄), evaporated, and purified on a silica gel column. Thedesired G-A dimer is eluted with CH₂Cl₂/CH₃OH (33/1). The appropriates fractionsare collected and evaporated to give a white foam. Yield: 95 mg, 0.1mmol, 48% with respect of 5'-OH-dA-3'-O-TBDM;³¹P NMR (CD₃CN) d 68.10,67.87; HPLC (spectrophotometrical purity at 260 nm = 80%).

Example 14

Synthesis of the dimer 5'-OH-dG^iBu-dC^Bz-3'-O-TBDMS cyanoethyl phosphorothioatetriester.

Coupling procedure of 5'-O-DMTr-dG^iBU-3'-cyanoethyl-phosphoramidite with5'-OH-dC^Bz-3'-O-TBDMS using the poly(4-vinylpyridinump-toluenesulfonate)(Aldrich):
5'-OH-dC^Bz-3'-O-TBDMS (100 mg, 0.22 mmol) and 5'-O-DMTr-dG^iBu-3'-cyanoethyl-phosphoramidite (371 mg, 0.45 mmol, 2 eq) are dissolved in anhydrousacetonitrile (15 ml). The solution is transferred under argon in a flaskcontaining the poly(4-vinylpyridinum p-toluenesulfonate) (690 mg, 2.3 mmoltos^-, 10.3 eq) and is shaken for 5 h. The reaction is followed by³¹P NMR. Theyield is determined by³¹P NMR. The desired dG-dC phosphite triester dimer isobtained with 100% of yield compared to the 5'-OH-dC^Bz-3'-O-TBDMS. Thecrude is a mixture of 5'-O-DMTr-dG^iBu-dC^Bz-3'-O-TBDMS cyanoethyl phosphitetriester (³¹P NMR (CD₃CN) (d 141.73, 141.26; 62.1%), 5'-O-DMTr-dG^iBu-3'-cyanoethylhydrogeno-phosphonate (d 9.05, 8.88; 37.9%).
Sulfurization: To a solution of 5'-O-DMTr-dG^iBu-dC^Bz-3'-O-TBDMS cyanoethylphosphite triester dimer (0.22 mmol) in anhydrous acetonitrile is addedAMBERLYST A26 tetrathionate form (0.65 g, 1.3 mmol S₄O₆^2-, 5.4 eq.). Thereaction mixture is shaken for 2 h. The reaction is followed by³¹P NMR. Theyield is determined by³¹P NMR. After filtration of the resins the desired dG-dCphosphorothioate triester dimer is obtained with 100% of yield compared tothe 5'-OH-dC^Bz-3'-O-TBDMS. The crude is a mixture of 5'-O-DMTr-dG^iBu-dC^Bz-3'-O-TBDMS cyanoethyl phosphorothioate (³¹P NMR (CD₃CN) (d 68.12, 67.73;61.2%), 5'-O-DMTr-dG^iBu-3'-cyanoethyl phosphorothioate diester (d 56.51,56.39; 25.4%), 5'-O-DMTr-dG^iBu-3'-cyanoethyl hydrogenophosphonate (d9.04, 8.85; 25.4%).³¹P NMR d.
Detritytlation: To a solution of 5'-O-DMTr-dG^iBu-dC^Bz-3'-O-TBDMS cyanoethylphosphorothioate triester (0.22 mmol) in 10 ml CH₂Cl₂/CH₃OH (7/3) is added0.5 ml (0.3 mmol, 1.4 eq.) of a solution of benzene sulfonic acid 10% inCH₂Cl₂/CH₃OH (7/3). The solution is stirred 1 h at 0°C. The reaction is washedwith 10 ml of a saturated solution of NaHCO₃, the organic layer is separated,dried (Na₂SO₄) and evaporated. The crude product is purified on a silica gelcolumn using CH₂Cl₂/CH₃OH (33:1). The appropriate fractions are collectedand evaporated to give a colorless oil. Yield: 99 mg, 0.1 mmol, 47%;³¹P NMR(CD₃CN) d 67.82, 67.56; MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M-H]⁺ m/z_exp = 914.78, m/z_calc = 914.03; HPLC(spectrophotometrical purity at 260 nm = 84%).

Example 15

Synthesis of 5'-O-DMTr-T-T-3'-O-DMTr-phosphorothioate diester.

To a solution of 5'-O-DMTr-T-T-3'-O-DMTrH-phosphonate diester (25 mg, 22mmol) in dichloromethane is added AMBERLYST A26 tetrathionate form (170mg, 0.29 mmol S₄O₆^2-, 13 eq.) and 0.1 mL triethylamine. The reaction mixtureis shaken for 78 h. The title compound was isolated after filtration of the resinand evaporation of the solvent. Yield: 28 mg, 22 mmol, 100%;³¹P NMR(CD₃CN) d 57.22; MALDI-TOF MS (negative mode, trihydroxy-acetophenoneas matrix) [M-H]^- m/z_exp = 1166.23, m/z_calc = 1666.25.

Example 16

Synthesis of 5'-O-DMTr-T-T-3'-O-TBDMS-phosphorothioate diester.

To a solution of 5'-O-DMTr-T-T-3'-O-TBDMSH-phosphonate (55 mg, 58 mmol)in dichloromethane is added AMBERLYST A26 tetrathionate form (250 mg,0.42 mmol S₄O₆^2-, 7.3 eq.) and 0.2 mL triethylamine. The reaction mixture isshaken for 26 h. The title compound was isolated after filtration of the resinand evaporation of the solvent. Yield: 63 mg, 58 mmol, 100%;³¹P NMR(CD₃CN) d 57.78, 57.72; MALDI-TOF MS (negative mode, trihydroxyacetophenoneas matrix) [M-H]^- m/z_exp = 977.04, m/z_calc = 977.13.

Example 17

Synthesis of AMBERLYST A26 tetrathionate form.

10 g commercial Amberlyst A26 hydroxide form (Rohm & Haas) is washedtwice with 20 mL methanol and twice with 20 mL dichloromethane and dried invacuum. Potassium tetrathionate (30.35 g, 100 mmol, 3 eq.) is dissolved in200 mL deionized water. The solution is added to the resin and shaken for 20hours. The solution is decanted of. The resin is washed with 4 L deionized water, twice with 100 mL methanol and twice with 100 mL dichloromethaneand dried under reduced pressure for 3 hours to give 8.5 g of solid-supportedtetrathionate. The reagents loading was determined by elemental analysis,giving a value of 23.25% for sulfur (4.24% for nitrogen, 45.74% for carbonand less than 100 ppm for potassium). Loading: 1.81 mmol S₄O₆^2- per gram ofresin.

Example 18

Synthesis of polymer-supported pyridinium

The commercially available strongly acidic ion-exchange resin DOWEX 50W X8H⁺ form (Fluka) is washed successively with water, HCl 2M, water until pH 7,methanol and dichloromethane to dry the resin. Then, the resin is stirred in asolution of pyridine 2M in acetonitrile or just washed with a slight flow of thesolution of pyridine 2M in acetonitrile for 15 minutes. Then, the resin is washedwith acetonitrile and dichloromethane and dried under vacuum over P₂O₅. Thereagents loading was determined by elemental analysis, giving a value of11.56% for sulfur and 3.97% for nitrogen. Loading: 2.83 mmol pyrH⁺ pergram of resin.

Example 19

Preparation of polystyrene-bound acid chloride

The commercial polystyrene-bound carboxy acid RAPP Polymere (5.0 g, 1.96mmol/g, 100-200 mesh, 1% DVB) is suspended in anhydrous CH₂Cl₂ (80 ml)and N,N-dimethylformamide (0.3 ml). Thionyl chloride (1.8 ml, 3.5 eq) areadded under stirring and the mixture is refluxed for 3h. The resin is filteredunder argon and washed with dried CH₂Cl₂ (100 ml), ether (100 ml) and driedunder vacuum for 4h.
IR (cm^-1): 1775 (C=O, Acid chloride)
Elemental analysis: Cl 7.43% (w/100g resin) (2.09 mmol/g)
Chloride titration: 2.1 mmol/g

Example 20

Synthesis of S'-O-DMTr-dA^Bz-dC^Bz-3'-O-Lev H-phosphonate

A solution of 5'-O-DMTr-dA^Bz-H-phosphonate TEA salt (123.4 mg, 0.150 mmol)and of 3'-O-Lev-dC^Bz (53.7 mg, 0.125 mmol) in 2.0 ml of CH₂Cl₂/py (1:1) isadded to polystyrene-bound acid chloride (388.8 mg, 2.1 mmol/g, 5.5 eq) thatis suspended in 2.5 ml of the same solvent. The mixture is shaken for 1h atroom temperature until the disappearance of the monomers. The reaction ismonitored by reverse phase HPLC. The resin is filtered, washed with CH₂Cl₂.The pyridinium salt present in solution is removed by aqueous extraction andthe aqueous phase is washed twice with CH₂Cl₂. The organic fractions are collected,dried over Na₂SO₄, the solvent is evaporated and the pyridine is eliminatedby coevaporation with toluene. The isolated product was dried undervacuum. Yield 89%.
³¹P NMR (CD₃CN) ä 10.03 ppm, 9.46 ppm.
MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp = 1134.13, m/z_calc = 1133.51.
The spectrophotometrical purity determined by HPLC is 93%.

Example 21

Synthesis of 5'-O-DMTr-dA^Bz-T-3'-O-Lev H-phosphonate

A solution of 5'-O-DMTr-dA^Bz-H-phosphonate TEA salt (123.4 mg, 0.150 mmol)and of 3'-O-Lev-T (42.5 mg, 0.125 mmol) in 2.0 ml of CH₂Cl₂/py (1:1) isadded to polystyrene-bound acid chloride (550.0 mg, 2.1 mmol/g, 7.7 eq) thatis suspended in 5.0 ml of the same solvent. The mixture is shaken for 30 minat room temperature until the disappearance of the monomers. The reaction is monitored by reverse phase HPLC. The resin is filtered, washed with CH₂Cl₂.The pyridinium salt present in solution is removed by aqueous extraction andthe aqueous phase is washed twice with CH₂Cl₂. The organic fractions are collected,dried over Na₂SO₄, the solvent is evaporated and the pyridine is eliminatedby coevaporation with toluene. The isolated product is dried under vacuum.Yield 88.5%
³¹P NMR (CD₃CN) ä 10.02 ppm, 9.08 ppm.
MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp= 1043.02, m/z_calc = 1045.00.
The spectrophotometrical purity determined by HPLC is 98%.

Example 22

Synthesis of 5'-O-DMTr-T-dC^Bz-3'-O-TBDMS H-phosphonate

A solution of 5'-O-DMTr-T-H-phosphonate TEA salt (106.4 mg, 0.150 mmol)and of 3'-O-TBDMS-dC^Bz (55.7 mg, 0.125 mmol) in 2.0 ml of CH₂Cl₂/py (1:1)is added to polystyrene-bound acid chloride (555.0 mg, 2.7 mmol/g, 10 eq)that is suspended in 5.0 ml of the same solvent. The mixture is shaken for 2hat room temperature until the disappearance of the monomers. The reaction ismonitored by reverse phase HPLC. The resin is filtered, washed with CH₂Cl₂.The pyridinium salt present in solution is removed by aqueous extraction andthe aqueous phase is washed twice with CH₂Cl₂. The organic fractions are collected,dried over Na₂SO₄, the solvent is evaporated and the pyridine is eliminatedby coevaporation with toluene. The isolated product is dried under vacuum.Yield 77.5%.
³¹P NMR (CD₃CN) ä 10.50 ppm, 10.00 ppm.
MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp = 1038.69, m/z_calc = 1037.20.
The spectrophotometrical purity determined by HPLC is 94%.

Example 23

Synthesis of 5'-O-DMTr -T-dC^Bz-3'-O-TBDMS phosphorothioate TEA salt

A solution of 5'-O-DMTr -T-dC^Bz-3'-O-TBDMSH-phosphonate (50 mg, 0.048mmol) in 5.0 ml of CH₂Cl₂ and 0.2 ml TEA is added to Amberlyst A26 tetrathionateform (141.0 mg, 1.7 mmol/g, 5 eq). The mixture is shaken overnight, the the resin is filtered and the solvent is evaporated. The product isdried under vacuum. Yield 100%.
³¹P NMR (CD₂Cl₂) ä 59.17 ppm, 58.99 ppm.
The spectrophotometrical purity determined by HPLC is 95%.

Example 24

Synthesis of 5'-O-DMTr-dA^Bz-T-3'-O-Lev phosphate TEA salt

A solution of 5'-O-DMTr-dA^Bz-T-3'-O-LevH-phosphonate (90 mg, 0.0863mmol) in 5.0 ml of CH₂Cl₂ and 0.2 ml TEA is added to(polystyrilmethyl)trimethylamonium metaperiodate (NOVABIOCHEM) (173.0mg, 2.5 mmol/g, 5 eq). The mixture is shaken over night, the resin is filteredand the solvent is evaporated. The product is dried under vacuum. Yield100%.
³¹P NMR (CD₂Cl₂) ä -1.37 ppm.
MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp= 1059.31, m/z_calc= 1060.03.
The spectrophotometrical purity determined by HPLC is 87%.

Example 25

Synthesis of 5'-O-DMTr-T-dA^Bz-dC^Bz-3'-O-Lev H-phosphonate

Detritylation of 5'-O-DMTr-dA^Bz-dC^Bz-3'-O-Lev H-phosphonate

TheH-phosphonate dimer 5'-O-DMTr-dA^Bz-dC^Bz-3'-O-Lev (120 mg, 0,106mmol) is dissolved in 4.0 ml of CH₂Cl₂/MeOH (7:3) and cooled in an ice bath.To this solution 1.0 ml of a solution of 10% BSA (benzene sulfonic acid) inCH₂Cl₂/MeOH (7:3) is added drop wise under stirring and the progress of thereaction is monitored by TLC. After 15 min the mixture is quenched with asolution of NaHCO₃. The organic layer is washed with water to remove anytrace of base, then it is dried over Na₂SO₄ and the solvent is evaporated. Theproduct is purified by precipitation from CH₂Cl₂ with ether and dried undervacuum. Yield 88%.
MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp= 830.75, m/z_calc = 831.75.
The spectrophotometrical purity determined by HPLC is 91%.

Coupling

A solution of 5'-O-DMTr-T-H-phosphonate TEA salt (93.8 mg, 0.132 mmol)and of 5'-OH-dA^Bz-dC^Bz-3'-O-LevH-phosphonate (73.2 mg, 0.088 mmol) in 2.0ml of CH₂Cl₂/py (1:1) is added to polystyrene-bound acid chloride (503.0 mg,2.1 mmol/g, 8 eq) that is suspended in 4.0 ml of the same solvent. The mixtureis shaken for 1h at room temperature until the disappearance of themonomers. The reaction is monitored by reverse phase HPLC. The resin isfiltered, washed with CH₂Cl₂. The pyridinium salt present in solution is removedby aqueous extraction and the aqueous phase is washed twice with CH₂Cl₂.The organic fractions are collected, dried over Na₂SO₄, the solvent is evaporatedand the pyridine is eliminated by coevaporation with toluene. The isolatedproduct is dried under vacuum. Yield 82%.
³¹P NMR (CD₂Cl₂) ä 10.23, 10.09, 9.70, 9.68, 9.52, 9.30, 9.24, 9.19 ppm.
MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp= 1421.06, m/z_calc = 1422.33.
The spectrophotometrical purity determined by HPLC is 82%.

Example 26

Synthesis of 5'-O-DMTr-dA^Bz-dA^Bz-T-3-O-Lev H-phosphonate

Detritylation of 5'-O-DMTr-dA^Bz-T-3'-O-Lev H-phosphonate

TheH-phosphonate dimer 5'-O-DMTr-dA^Bz-T-3'-O-Lev (105 mg, 0,100 mmol)is dissolved in 4.0 ml of CH₂Cl₂/MeOH (7:3) and cooled in an ice bath. 1.0 mlof a solution of 10% BSA in CH₂Cl₂/MeOH (7:3) is added drop wise under stirringand the progress of the reaction is monitored by TLC. After 15 min themixture is quenched with a solution of NaHCO₃. The organic layer is washedwith water to remove any trace of base, then it is dried over Na₂SO₄ and thesolvent is evaporated. The product is purified by precipitation from CH₂Cl₂ inether and dried under vacuum. Yield 70%.
MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp = 742.25, m/z_calc = 742.66.
The spectrophotometrical purity determined by HPLC is 92%.

Coupling

A solution of 5'-O-DMTr-dA^Bz-H-phosphonate TEA salt (69.8 mg, 0.084 mmol)and of 5'-OH-dA^Bz-dT-3'-O-LevH-phosphonate (52.2 mg, 0.070 mmol) in 2.0ml of CH₂Cl₂/py (1:1) is added to polystyrene-bound acid chloride (311.0 mg,2.1 mmol/g, 7.7 eq) that is suspended in 2.0 ml of the same solvent. Themixture is shaken for 3h at room temperature until the disappearance of themonomers. The reaction is monitored by reverse phase HPLC. The resin isfiltered, washed with CH₂Cl₂. The pyridinium salt present in solution is removedby aqueous extraction and the aqueous phase is washed twice with CH₂Cl₂.
The organic fractions are collected, dried over Na₂SO₄, the solvent is evaporatedand the pyridine is eliminated by coevaporation with toluene. The isolatedproduct is dried under vacuum. Yield 75%.
³¹P NMR (CD₂Cl₂) ä 10.09, 9.39, 8.82, 8.76, 8.30, 7.56 ppm.
MALDI-TOF MS (positive mode, trihydroxyacetophenone as matrix) [M+H]⁺m/z_exp= 1445.60, m/z_calc = 1447.40.
The spectrophotometrical purity determined by HPLC is 91.5%.

Claims

A method for preparing an oligonucleotide comprising the steps of
a) providing a 3'-protected compound having the formula:
wherein
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylen linkage
R₃ is a hydroxyl protecting group, a 3'-protected nucleotide or a 3'-protectedoligonucleotide
b) reacting said compound with a nucleotide derivative having a 5'-proctectiongroup in the presence of a solid supported activator to give anelongated oligonucleotide with a P(III)-internucleotide bond
c) processing the elongated oligonucleotide with a P(III)-internucleotidebond by steps c1) and c2) in any sequence
c1) capping by reacting with a solid supported capping agent
c2) oxidizing by reacting the oligonucleotide with a solid supported oxidizingreagent
d) removing the 5'-protection group by treatment with a solid supportedagent or removing the 5'-protection group with a removal agent followed by addition of a solid supported scavenger or followed by extraction.
The method of claim 1, wherein the nucleotide derivative having a 5'-proctectiongroup of step b) has the following formula:
wherein
X is a P(III)-function
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylen linkage
R₅ is a hydroxyl protecting group, a 5'-protected nucleotide or a 5'-protectedoligonucleotide.
A method for preparing an oligonucleotide comprising the steps of
a) providing a 5'-protected compound having the formula:
wherein
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylen linkage
R₅ is a hydroxyl protecting group, a 5'-protected nucleotide or a 5'-protectedoligonucleotide
b) reacting said compound with a nucleotide derivative having a 3'-proctectiongroup in the presence of a solid supported activator to give anelongated oligonucleotide with a P(III)-internucleotide bond
c) processing the elongated oligonucleotide with a P(III)-internucleotidebond by steps c1) and c2) in any sequence
c1) capping by reacting with a solid supported capping agent
c2) oxidizing by reacting the oligonucleotide with a solid supported oxidizingreagent
d) removing the 3'-protection group by treatment with a solid supportedagent or removing the 3' -protection group with a removal agent followedby addition of a solid supported scaenger or followed by extraction.
The method of claim 3, wherein the nucleotide derivative having a 3'-proctectiongroup has the following formula:
wherein
X is a P(III)-function
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylen linkage
R₅ is a hydroxyl protecting group, a 5'-protected nucleotide or a 5'-protectedoligonucleotide
The method of any one of claims 1 to 4, comprising the further step of e)repeating steps a) to d) at least once.
The method of any one of claims 1 to 5, wherein the nucleotide derivative ofstep b) is a phosphoramidite or a H-phosphonate.
The method of any one of steps 1 to 6, wherein the solid supported activatorof step b) is selected from the group consisting of a solid support bearing apyridinium salt, a cation exchange solid support with an optionally substitutedpyridinium, or a cation exchange solid support with an optionally substitutedimidazolium salt, a solid support bearing an optionally substitutedazole (imidazol, triazole, tetrazole), a salt of a weak base anion exchangeresin with a strong acid, a weak cation exchange resin (carboxylic) in itsprotonated form, a solid support bearing an optionally substituted phenol, asolid support bearing a carboxylic acid chloride/bromide, a sulfonic acid chloride/bromide,a chloroformate, a bromoformate, a chlorosulfite, a bromosulfite,a phosphorochloridate and a phosphorbromidate.
The method of any one of claims 1 to 7, wherein the solid supported oxidizingreagent is selected from the group consisting of solid supported periodates,permanganates, osmium tetroxides, dichromates, hydroperoxides,substituted alkylamine oxides, percarboxylic acid and persulfonic acid.
The method of any one of claims 1 to 8, wherein the oxidizing is a sulfurization.
The method of claim 9, wherein the solid supported oxidizing reagent isselected from the group consisting of a solid supported tetrathionate, a solid supported alkyl or aryl sulfonyl disulfide, a solid supported optionally substituteddibenzoyl tetrasulfide, a solid supported bis(akyloxythio-carbonyl)tetrasulfide,a solid supported optionally substituted phenylacetyldisulfide, a solid supported N-[(alkyl or aryl)sulfanyl] alkyl or aryl substitutedsuccinimide and a solid supported (2-pyridinyldithio) alkyl or aryl.
The method of any one of claims 1 to 10, wherein the solid supported cappingagent is a solid supported activated acid, preferably a carboxylic acidchloride, carboxylic acid bromide, azolide, substituted azolide, anhydride orchloroformate or phosphorochloridate, or a solid supported phosphoramidite,or a solid supported H-phosphonate monoester.
The method of any one of claims 1 to 11, wherein the 5'-protection is adimethoxytrityl group (DMTr) or a monomethoxytrityl group (MMTr) and thesolid supported agent of step d) is an cationic ion exchanger resin in the H⁺form or ceric ammonium nitrate.
The method of any one of claims 1 to 12, wherein the 3'-protection is a silylgroup and the solid supported agent of step d) is an anionic ion exchangerresin in the F-form.
Solid supported sulfurization agent consisting of solid supported amine and atetrathionate having the formula S₄O₆.
A method for coupling a compound having the formula
wherein
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylen linkage
R₃ is a hydroxyl protecting group, a nucleotide or an oligonucleotide
with a nucleotide derivative having a 5'-proctection group in the presence ofa solid supported activator to give an elongated oligonucleotide with aP(III)-internucleotide bond
A method for coupling a compound having the formula
wherein
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylen linkage
R₅ is a hydroxyl protecting group, a 5'-protected nucleotide or a 5'-protectedoligonucleotide
with a nucleotide derivative having a 3'-proctection group in the presence ofa solid supported activator to give an elongated oligonucleotide with aP(III)-internucleotide bond.
A method for preparing an oligonucleotide comprising the steps of
a) providing a compound having the formula:
wherein
B is a heterocyclic base
R₂ is H, a protected 2'-hydroxyl group, F, a protected amino group, an O-alkylgroup, an O-substituted alkyl, a substituted alkylamino or a C4'-O2'methylen linkage
and
R₃ is a hydroxyl protecting group, a protected nucleotide or a protectedoligonucleotide and R₅ is a P(III) function
or
R₅ is a hydroxyl protecting group, a protected nucleotide or a protectedoligonucleotide and R₃ is a P(III) function
b) reacting said compound with a nucleotide derivative having a 3' or 5'-freeOH-group in the presence of a solid supported activator to give an elongatedoligonucleotide with a P(III)-internucleotide bond
c) processing the elongated oligonucleotide with a P(III)-internucleotidebond by steps c1) and c2) in any sequence
c1) capping by reacting with a solid supported capping agent
c2) oxidizing by reacting the oligonucleotide with a solid supported oxidizing reagent
d) removing the 3' or 5'-protection group by treatment with a solid supportedagent or removing the 3' or 5'-protection group with a removalagent followed by addition of a solid supported scavenger or followed byextraction.